Sustained release of apo a-i mimetic peptides and methods of treatment

ABSTRACT

A method including advancing a delivery device through a lumen of a blood vessel to a particular region in the blood vessel; and introducing a composition including a sustained-release carrier and an apolipoprotein A-I (apo A-I) synthetic mimetic peptide into a wall of the blood vessel at the particular region or a perivascular site, wherein the peptide has a property that renders the peptide effective in reverse cholesterol transport. A composition including an apolipoprotein A-I (apo A-I) synthetic peptide, or combination of an apo A-I synthetic mimetic peptide and an Acyl CoA cholesterol: acyltransferase (ACAT) inhibitor in a form suitable for delivery into a blood vessel, the peptide including an amino acid sequence in an order reverse to an order of various apo A-I mimetic peptides, or endogenous apo A-I analogs, or a chimera of helix 1 and helix 9 of endogenous apo A-I.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 11/858,862 filed Sep. 20, 2007.

SEQUENCE LISTING

A paper copy of the Sequence Listing entitled “5618USP1” is hereinincorporated by reference. This Sequence Listing consists of SEQ. IDNOs:1-12.

FIELD OF THE INVENTION

Compositions and methods for facilitating reverse cholesterol transport.

BACKGROUND OF THE INVENTION

Cholesterol is a major component of atherosclerotic plaque. Cholesterolaccumulation within atherosclerotic plaque occurs when cholesterolinflux into an arterial wall exceeds cholesterol efflux. Increasedinflux of cholesterol into the arterial wall is accompanied by anincreased influx of monocytes/macrophages, which absorb oxidizedaggregated low density lipoproteins (LDL) and store the cholesterolesters.

Current strategies to reduce coronary heart disease are primarilydirected at reducing the influx of cholesterol into the arterial wall bylowering LDL cholesterol concentration. While lowering of plasma LDLlevels offers some protection from coronary heart disease, theprotection is not complete. As a result, there is an interest instrategies aimed at enhancing cholesterol efflux from the arterial walland promoting its transport to the liver for excretion.

Cholesterol circulating in the blood is carried by plasma lipoproteins.Plasma lipoproteins are classified into groups according to size. Ofthese, the low density lipoprotein (LDL) and high density lipoprotein(HDL) are primarily the major cholesterol carrier proteins. The proteincomponent of LDL, apolipoprotein B (Apo B), constitutes the atherogeniccomponent. Apo B is not present in HDL. HDL includes apolipoprotein A-I(apo A-I) and apolipoprotein A-II (Apo A-II) as well as otherapolipoproteins.

Various forms of HDL have been described on the basis of electrophoreticmobility and include two major fractions: a first fraction with α-HDLmobility and another fraction referred to as pre-β HDL. Pre-β HDL isthought to be the most efficient HDL subclass for inducing cellularcholesterol efflux. Pre-β HDL fractions includes apo A-I, phospholipidsand free cholesterol. Pre-β HDL are considered to be acceptors ofcellular free cholesterol and are believed to transfer free andesterified cholesterol to α-HDL.

Two pathways have been proposed to describe the movement of cholesterolfrom a plasma membrane to acceptor particles such as pre-β HDL. In the“aqueous diffusion pathway,” cholesterol molecules spontaneously desorbfrom cell membranes and are then incorporated into acceptor particles(pre-β HDL) after traversing the intervening aqueous space by diffusion.It is believed that the aqueous diffusion pathway does not requireinteraction with specific cell receptors.

The second model, referred to as the “microsolubilization pathway,”involves the interaction of HDL (presumably an apo A-I interaction) witha cell surface binding site. The HDL induces an intracellular signalleading to translocation of cholesterol from intracellular sites to theplasma membrane. The physiological acceptors or carriers for thetranslocated cholesterol are nascent HDL particles, including α-HDL andpre-β HDL.

Cholesterol that is transferred to nascent HDL particles is esterifiedby lecithin-cholesterol acyl transferase (LCAT) to cholesteryl esters.These esters are hydrophobic and tend to move into the core of the HDLparticle. The HDL cholesteryl esters may return or be delivered to theliver and are excreted from the liver into bile, either directly orafter conversion to bile cells.

It is believed that α-HDL and pre-βHDL particles, the primary acceptorsor carriers for translocated cholesterol, do not occur in the samerelative fractions as nascent HDL particle in the blood stream of anadult human. Thus, the carrier potential of each fraction is believed tobe inversely proportional to its relative fraction of the total HDLquantity. In other words, the fraction with the highest carrierpotential (pre-β HDL) occurs in the smallest overall amount in vivo.

SUMMARY OF THE INVENTION

An embodiment of a composition comprising an apolipoprotein A-I (apoA-I) synthetic mimetic peptide complexed with phospholipid; and acarrier associated with the apo A-I synthetic mimetic peptide whereinthe carrier is selected from the group consisting of a liposome, apolymerosome, a micelle, a particle, a gel, a microbubble, aprecipitated peptide particle, a porous glass particle, and a rod isherein disclosed. An embodiment of a composition comprising asustained-release carrier; and at least one of a non-complexedapolipoprotein A-I (apo A-I) synthetic mimetic peptide or a phospholipidcapable of complexing with apo A-I synthetic mimetic peptideencapsulated within the sustained-release carrier wherein thesustained-release carrier is suspended within a solution to form adispersion is herein disclosed.

An embodiment of a method comprising stabilizing an apolipoprotein A-I(apo A-I) synthetic mimetic peptide in solution wherein stabilizingcomprises one of: (i) complexing the apo A-I synthetic mimetic peptidewith a phospholipid; (ii) increasing a concentration of the apo A-Isynthetic mimetic peptide; or (iii) a combination thereof is hereindisclosed.

An embodiment of a method of fabrication comprising adding a solutioncontaining an apolipoprotein A-I (apo A-I) synthetic mimetic peptide toa solution containing a phospholipid resulting in a combination; andsubjecting the combination to a mixing process is herein disclosed. Anembodiment of a method of fabrication comprising fabricating asustained-release carrier encapsulating at least one of a non-complexedapo A-I synthetic mimetic peptide or a phospholipid capable ofcomplexing with apo A-I synthetic mimetic peptide; and suspending thesustained-release carrier in a solution wherein the suspension issuitable for injecting into a treatment site suitable for reversecholesterol efflux is herein disclosed.

An embodiment of a method of treatment comprising forming a mimeticpeptide/phospholipid complex at a treatment site in situ wherein formingcomprises locally delivering a non-complexed mimetic peptide and aphospholipid capable of complexing with the non-complexed mimeticpeptide to the treatment site wherein at least one of the non-complexedmimetic peptide and the phospholipid are encapsulated within at leastone sustained-release carrier is herein disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the turbidity change of liposome (multilamellar vesicles,MLV) after mixing with a peptide.

FIG. 1B shows charts (i) through (vi) illustrating circular dichromism(CD) spectrums of both a solution including a mimetic peptide, i.e., SEQID NO: 4, (charts (i) through (iii)) and a solution including a mimeticpeptide/phospholipid complex, i.e., SEQ ID NO: 4 complexed with DMPC(charts (iv) through (vi)), as their concentrations are increased.

FIG. 1C shows charts (vii) through (x) illustrating CD spectrums of asolution including a mimetic peptide, i.e., SEQ ID NO: 4, after heatingthen cooling at different concentrations.

FIG. 1D shows charts (xi) and (xii) illustrating time course and doseresponse of a mimetic peptide, i.e., SEQ ID NO: 4, on the reversedcholesterol transport in THP-1 cells and J774 cells loaded with AcLDL orOxLDL.

FIG. 1E shows a chart illustrating the predictive values of treatmentagent release from both monodisperse and polydisperse nanoparticles.

FIG. 1F shows chart (xiii) illustrating release profiles over days for1.2 μm average size microparticles and 1.8 μm average sizemicroparticles and chart (xiv) illustrating a release profiles over daysfor a 1:1 ratio of 1.2 μm average size microparticles and 1.8 μm averagesize microparticles.

FIG. 1 is a simplified cross-sectional view of an embodiment of asubstance delivery apparatus in the form of a catheter assembly having aballoon and a therapeutic substance delivery assembly.

FIG. 2 schematically illustrates a portion of coronary artery networkhaving a catheter assembly introduced therein.

FIG. 3 presents a block diagram for introducing a treatment composition.

FIG. 4 schematically illustrates a portion of a coronary artery networkhaving a stent placed therein and a catheter assembly introducedtherein.

FIG. 5 schematically illustrates a simplified cross-sectional view of asecond embodiment of a substance delivery apparatus in the form of acatheter assembly having a balloon and a treatment agent deliveryassembly.

FIG. 6 shows the blood vessel of FIG. 4 and a second embodiment of acatheter assembly to deliver a treatment agent introduced into the bloodvessel.

FIG. 7 shows the blood vessel of FIG. 4 and a fourth embodiment of acatheter assembly to delivery a treatment agent introduced into theblood vessel.

FIG. 8 shows the blood vessel of FIG. 4 and a fifth embodiment of acatheter assembly to deliver a treatment agent introduced into the bloodvessel.

DETAILED DESCRIPTION OF THE INVENTION

The following embodiments describe techniques, compositions and devicesdirected, in one aspect, at improving reverse cholesterol transport bythe aqueous diffusion pathway or the microsolubilization pathway. Directvascular protective effects of HDL have been attributed to apo A-I orapo A-I-associated molecules in HDL. Amphipathic helical peptides thatmimic the actions of apo A-I have been shown to have anti-atherogeniceffects in animal models.

Endogenous apo A-I molecule (human) is a single polypeptide chain with243 amino acids consisting of ten 22-mer amphipathic α-helices intandem. The majority of the α-helices, i.e., helices 1, 2 and 5-8 areclass A helices, while the remainder, i.e., 3-4, 9-10 are class Yhelices. Endogenous apo A-I contains a globular N-terminal domain(residues 1 to 43) and a lipid-binding C-terminal domain (residues 44 to243). Endogenous apo A-I is synthesized by the liver and small intestineas a preproprotein (260 amino acid residues) which is secreted as aproprotein (249 amino acid residues) that is rapidly cleaved to generatea mature polypeptide having 243 amino acid residues.

Treatment Agents

In one embodiment, a synthetic apo A-I mimetic peptide (hereinafter,referred to as a “mimetic peptide”) combined with a carrier is locallydelivered. “Local” means that the mimetic peptide is delivered to a siteof dimensions less than 40 mm. For example, “local delivery” includesdelivery into a vessel wall, peri-vascular arterial site, or a lesionassociated with a vessel wall such as a coronary vessel wall as opposedto delivery into the systemic circulation. “System delivery” includesdelivery to multiple organs or the entire body by, for example, anintravenous injection in the arm. Local delivery may provide enhanceddelivery efficiency and minimize treatment agent (e.g., synthetic apoA-I mimetic peptide) loss into the systemic circulation, therebyallowing application of lower doses and longer duration of activity.Local delivery also improves overall effectiveness in modulatingcoronary arterial response to injury.

According to some embodiments, a synthetic apo A-I mimetic peptide“mimics” endogenous human apo A-I in the sense that the mimetic peptideis capable of the removal of cholesterol, i.e., reverse cholesteroltransport or efflux. In one embodiment, the mimetic peptide includes atleast one class A amphipathic α-helix having positively charged aminoacid residues clustered at a hydrophobic-hydrophilic interface andnegatively-charged amino acid residues clustered at a center of ahydrophilic face. Other characteristic properties of apo A-I mimeticpeptides include peptides with a non-polar side of aromatic amino acids,like phenylalanine or tyrosine, and positively-charged amino acids(e.g., glutamic acid) between two α-helices having a suitable distance(e.g., approximately 3.6 amino acid residues).

Examples of suitable synthetic apo A-I mimetic peptides that may belocally delivered include, but are not limited to:

-   -   (i) An 18 amino acid peptide 4F (L-4F, D-4F), DWFKAFYDKVAEKFKEAF        (SEQ ID NO: 1), and its homologs, derivatives and analogs;    -   (ii) An 18 amino acid peptide, DWLKAFYDKVAEKLKEAF (SEQ ID NO:        2), and its homologs, derivatives and analogs; and    -   (iii) A 33 amino acids peptide,        PALEDLRQGLLPVLESFKVFLSALEEYTKKLNTQ (SEQ ID NO: 3), and its        homologs, derivatives and analogs.

As described, the 18-mer and 33-mer peptides begin at the amino end of apolypeptide chain (e.g., the peptide chain is read left to rightstarting with the amino-terminal residue). One of ordinary skill in theart would appreciate that “D” and “L” designations refer to theenantiomers of a compound based on the actual geometry of eachenantiomer.

Other amphipathic helix peptides, such as Apo A-II and Apo J peptide,and the homologs, derivatives and analogs thereof are also suitable.Peptides may consist of D amino acids, L amino acids, a racemic backboneof D and L amino acids, or any mixture thereof. The N-terminal may bemodified by components including, but not limited to, acetyl groups, andC-terminal carboxyl may be modified by components, including but notlimited to, amides or esters. These modified peptide structures mayprovide protection against premature degradation. Examples of modifiedpeptides include, but are not limited to:

-   -   (iv) An 18-mer peptide, 4F: Ac-DWFKAFYDKVAEKFKEAF-NH₂ (SEQ ID        NO: 4); and    -   (v) A 33-mer peptide, helices 9/10:        Ac-PALEDLRQGLLPVLESFKVFLSALEEYTKKLNTQ-NH₂ (SEQ ID NO: 5).

The amino acids represented by letters in the above examples andthroughout this description are as follows:

A Ala Alanine C Cys Cysteine D Asp Aspartic acid E Glu Glutamic acid FPhe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K LysLysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline QGln Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine WTrp Tryptophan Y Tyr Tyrosine

The 18-mer D-4F peptide mimetic has been shown to be effective inreverse cholesterol transport without a lipid complex. However, thispeptide may be administered in the form of a phospholipid complex as canother peptides described herein. Examples of phospholipids which may becomplexed with peptide mimetics in accordance with embodiments of theinvention include, but are not limited to,dimyristoylphosphatidylcholine (DMPC), dipalmitoyl phosphatidylethanolamine (DPPE), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1-dalmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Eggphosphatidylcholine (EPC), hydrogenated egg phosphatidylcholine (HEPC),soybean phosphatidylcholine (SPC), hydrogenated soybeanphosphatidylcholine (HSPC). Incompletely lipidated endogenous apo A-I,existing as a flexible conformation or molten globular state, is knownto readily associate with lipids due to its amphipathic α-helicalsegments. Thus, mimetic peptides according to embodiments of theinvention are expected to similarly associate with lipids due toinclusion of at least one amphipathic α-helical segment. In oneembodiment, a synthetic apo A-I mimetic peptide may be formulated as acomplex with a phospholipid such as dimyristoyl phosphocholine (DMPC) toform a lipid complex. Both 18-mer L-4F and D-4F mimetic peptides havebeen shown to function more effectively in reverse cholesterol transportin vitro when a phospholipid complex is presented. The 33-mer peptidemimetic, in particular, requires phospholipid complex formation foreffective reverse cholesterol transport.

As noted above, endogenous apo A-I molecule (human) is a singlepolypeptide chain with 243 amino acids consisting of 10 amphipathicα-helices. One unit of helix turns consistently and includes 3.6 aminoacid residues. Therefore, even though a peptide sequence is reversed, arelative location of hydrophobic and hydrophilic side chains is similarto the original peptide when peptides form α-helix structure. Thus, inanother embodiment, a suitable apo A-I mimetic peptide foratherosclerosis treatment (e.g., reverse cholesterol transport) is apeptide including amino acids arranged in an order reverse to the orderof an endogenous apo A-I peptide or a portion thereof.

Examples of reverse sequence synthetic apo A-I mimetic peptides (withoptionally modified N— and C-terminals) include, but are not limited to:

(SEQ ID NO: 6) (vi) Ac-FAEKFKEAVKDYFAKFWD-NH₂ (4F); (vii) (SEQ ID NO: 7)Ac-FAEKLKEAVKDYFAKLWD-NH₂; and (viii) (SEQ ID NO: 8)Ac-NLKKTYEELASLFSVKFSELVPLLGQRLDELAP-NH₂.

The reverse sequence apo A-I peptides may be formulated as phospholipidcomplexes and/or prepared as a treatment composition with, for example,a buffer as described above.

Work with the secondary structure of apo A-I has identified helix 1 ashaving high lipid binding affinity. The chimera of helices 1 and 9 hasdemonstrated high hydrophobicity and an acceptable phospholipidarrangement (e.g., DMPC clearance). Thus, in one embodiment, a suitableapo A-I synthetic peptide mimetic for atherosclerosis therapy is achimera of helices 1 and 9. The peptides may be represented as follows:

(iv) 1/9 chimera: (SEQ ID NO: 9)Ac-LKLLDNWDSVTSTFSKLREQLGPALEDLRQGLL-NH₂; and (x) 9/1 chimera: (SEQ IDNO: 10) Ac-PALEDLRQGLLPKLLDNWDSVTSTFSKLREQLG-NH₂.In this context, a chimera is a recombinant DNA molecule containingunrelated genes in the sense that the genes are each not a component ofthe same α-helix of the 10 α-helices of endogenous apo A-I.

In the embodiments of the chimera of helices 1 and 9 described above,the peptides may be modified (as shown) with an N-terminal acetyl groupand a C-terminal amide or ester to stabilize the amphipathic nature ofthe helices. The peptides may further be formulated as phospholipidcomplexes with, for example, DMPC, to act as an acceptor for cholesteroland/or prepared as a treatment composition with, for example, a buffer.The peptides may be synthesized or fabricated by recombinant methodswith L- or D-amino acids. Still further, the reversed sequences shouldhave similar potency.

EXAMPLE 1

The mimetic peptides described here can be synthesized by bothdi-tert-butyldicarbonate (boc)-, andN-α-(9-fluorenylmethoxycarbonyl)-N-γ-trityl-L-asparagine (Fmoc)-basedsolid phase synthesis. Described herein is Fmoc-based solid phasepeptide synthesis. Rink amide resins, for example, Rink amide MBHAresin, or Fmoc-PAL-PEG-PS resin are used to install carboxyamides at theC-terminus of the synthesized peptides. For peptides with unmodifiedC-terminus, Wang resin is used for solid phase peptide synthesis (SPSS).Any other resins for SPSS can be used to synthesize peptides accordingto embodiments of the invention. Upon completion of peptide synthesis,if necessary, the peptide N-terminus is modified by treatment with, forexample, acetic anhydride (10 eq.) and diisopropylethylamine (DIPEA) (10eq.), or any carboxylic acid derivatives (2-5 eq.) inN,N-dimethylformamide (DMF)/dichloromethane (DCM) (1:1, v/v) withcoupling reagent (2-5 eq.) and base (2-5 eq.). Side chain protectiongroups were removed and the peptide is simultaneously cleaved from theresin with cleavage cocktails, such as Reagent K, or 94% trifluoroaceticacid (TFA), 2.5% water, 2.5% ethanedithiol (EDT), and 1%triisopropylsilane (TIS) for cysteine-containing peptide, or any otherappropriate cleavage cocktails. Purification of peptides was performedby preparative high performance liquid chromatography (HPLC) or liquidchromatography/mass spectrometry (LC/MS) with a water/acetonitrilegradient containing 0.1% TFA or formic acid. When necessary, counter ioncan be exchanged to another acid, such as acetic acid. For amino acidcoupling, the following coupling reagent can be used (but other reagentscan also be used for peptide synthesis): N,N′-dicyclohexyl-carbodiimide(DCC), benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP),benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP), (1H-Benzotriazol-1-yl)1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU),1H-Benzotriazolium-1-[bis(dimethylamino)methylene]tetrafluoroborate-(1,3)-oxide(TBTU), 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TNTU),O—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TSTU), and bromo-tris-pyrrolidino phosphoniumhexafluorophosphate(PyBrOP). 1-Hydroxybenzotriazole anhydrous (HOBt) can be added to thereaction mixture to prevent side reaction and act as catalyst.

In some embodiments, secondary structures of mimetic peptides inaccordance with embodiments of the invention can be stabilized bysubjecting the mimetic peptide to methods such as: complexing themimetic peptide with a phospholipid; increasing a concentration of themimetic peptide in aqueous medium; or any combination thereof.Stabilization of the secondary structure of the mimetic peptide can beuseful in order to retain a functional property of the mimetic peptide.

In one embodiment, a mimetic peptide, e.g., SEQ ID NO: 4, can be mixedwith multilamellar vesicles (MLV) comprising DMPC to form a mimeticpeptide/phospholipid complex for stabilization thereof. In someembodiments, stabilization of a mimetic peptide/phospholipid complex iscorrelated with efficacy of reverse cholesterol efflux in vitro. Themimetic peptide/phospholipid complex acts not only as a stabilizer forthe secondary structure, but also as an acceptor for the freecholesterol circulating throughout the vasculature. In some embodiments,the mimetic peptide:phospholipid ratio can be between 1:2.5 and 1:5 w/w.In some embodiments, the mimetic peptide/phospholipid complex can rangebetween about 4 nanometers and about 14 nanometers, on average about 10nanometers. Some complex entities aggregate to form aggregates in therange between about 70 nanometers and 100 nanometers, on average about80 nanometers.

EXAMPLE 2

A mimetic peptide, SEQ ID NO: 4, was mixed with multilamellar vesiclescomprised of DMPC. A DMPC chloroform solution was gently dried bynitrogen stream in the fume hood or by rotor evaporator. Resultant lipidfilm was further dried in vacuum desiccators and subjected to vacuum forat least 3 hours to remove residual chloroform. Phosphate bufferedsaline (PBS) was added to the lipid film and mixed using a vortex mixerto prepare an MLV suspension (2.5 mg/mL). The peptide was dissolved inPBS (1 mg/mL) then mixed with the same volume of MLV suspension at roomtemperature. FIG. 1A shows a chart illustrating the turbidity change ofthe resultant complex. At 0.5 mg/mL mimetic peptide in solution, theabsorbance at 400 nm is zero. At 1.25 mg/mL DMPC in solution, theabsorbance at 400 nm is 0.1. When the two solutions are combined andafter approximately 5 minutes of mixing, the absorbance begins toapproach zero. After approximately 15 minutes of mixing, the solutionbecomes completely clear, i.e., the absorbance is zero, thus evidencingmimetic peptide/phospholipid complexing.

Stabilization has been shown to increase upon an increase inconcentration of the mimetic peptide or the mimetic peptide/phospholipidcomplex. FIG. 1B shows charts (i) through (vi) illustrating circulardichromism (CD) spectrums and a summary table of calculated percentsecondary structure of both a solution including a mimetic peptide,i.e., SEQ ID NO: 4, (charts (i) through (iii)) and a solution includinga mimetic peptide/phospholipid complex, i.e., SEQ ID NO: 4 complexedwith DMPC (charts (iv) through (vi)), as their concentrations areincreased. As the concentration of the mimetic peptide as shown in thesummary table of FIG. 1B increases from 0.125 mg/mL mimetic peptide to0.5 mg/mL mimetic peptide, the alpha helicity increases from 10.5% to19.5%, thus indicating increasing stabilization of the alpha-helix.Similarly, as the formation of the mimetic peptide/phospholipid complexas shown in the summary table of FIG. 1B increases, the alpha helicityincreases from 10.5% to 33% at 0.125 mg/mL mimetic peptide, from 16% to36% at 0.25 mg/mL mimetic peptide, and from 19.5% to 34% at 0.5 mg/mLmimetic peptide, thus indicating increasing stabilization of thealpha-helix.

Secondary structure has been shown to be maintained when a mimeticpeptide is subjected to heat and then cooled. FIG. 1C shows charts (vii)through (x) illustrating CD spectrums of a solution including a mimeticpeptide, i.e., SEQ ID NO: 4, after heating then cooling at differentconcentrations. As shown in each chart (vii) through (x), a CD spectrumof the mimetic peptide was obtained at t₁=25° C., then heated to t₂=70°C., then cooled back to t₃=25° C. for varying concentrations, e.g., 0.5mg/mL mimetic peptide, 0.25 mg/mL mimetic peptide, 0.125 mg/mL mimeticpeptide, and 0.0625 mg/mL mimetic peptide. The spectrums at t₁ and t₃show a substantially identical spectrum for each of t₁ and t₃ at thevarious concentrations, thus indicating the mimetic peptide's secondarystructure change is reversible when subjected to heat.

FIG. 1D shows charts (xi) and (xii) illustrating time course and doseresponse of a mimetic peptide, i.e., SEQ ID NO: 4, on the reversedcholesterol transport in THP-1 cells (a human monocyte-derived cellline) and J774 cells, respectively, loaded with AcLDL or OxLDL. At alltested concentrations, the mimetic peptide/phospholipid complex removescholesterol more effectively than the mimetic peptide alone.

Carriers

According to embodiments of the invention, a delivery compositionincluding a synthetic apo A-I mimetic peptide also includes a carrier.In one embodiment, the apo A-I mimetic peptide can be encapsulated,suspended, disposed within or on (chemisorbed), or loaded into(hereinafter, collectively referred to as “associated with”) abiodegradable carrier. One of ordinary skill in the art would know thatthe amount of mimetic peptide is given in various dosages, includingmaximum and minimum amounts. Examples of biodegradable carriers include,but are not limited to, a liposome, a polymerosome, a micelle, aparticle, a microbubble, and a gel. Examples of particles include, butare not limited to, microparticles, nanoparticles, and core-shellparticles. In some embodiments, the biodegradable carrier is formulatedsuch that it is bioerodable when present in physiological conditions.Example 2 above may be considered an exemplary example of a mimeticpeptide, i.e., SEQ ID NO: 4, associated with a carrier, i.e., DMPCmultilamellar vesicles, or liposomes.

In one embodiment, the biodegradable carrier for a synthetic apo A-Imimetic peptide is a liposome. “Liposomes” are artificial vesicles thatare approximately spherical in shape and can be produced from naturalphospholipids, sphingolipids, ceramides, cholesterol or estradiol.Generally, a liposome has a lipid bilayer membrane encapsulating anaqueous solution, i.e., “core.” The lipid bilayer membrane allows forfusion with an endogenous (or exogenous) cell membrane, which, similarto the liposome, comprises a semipermeable lipid bilayer. In oneexample, the peptide mimetic may be included in the lipid bilayer of theliposome (as opposed to within the core). This can be achieved eitherduring liposome formation or in a post-insertion method. It isanticipated that such an embodiment will provide simultaneous extractionof cholesterol from a vulnerable plaque lesion and a potential foralteration of liposomal structure rigidity resulting in altered masstransport properties.

In one method, phospholipids and synthetic apo A-I mimetic peptide aremixed with estradiol in chloroform. Suitable phospholipids include, butare not limited to, DMPC, DPPE, DLPC, DMPC, DPPC, DOPC, POPC, EPC, HEPC,SPC, and HSPC. The liposomes may also be hydrophilically modified bycoating with an agent such as poly(ethylene glycol) or dextran. Suchcoating tends to avoid detection from the body's immune system. Aftermixing, the solvent (and optional co-solvent) can be evaporated withheat or ambient temperature in a round bottom flask. Resultant lipidswill be deposited on the glass surface. The deposited lipid film will bere-suspended in aqueous solution to form multilamellar (or unilamellar)vesicles, and extruded to prepare appropriate sized liposomes. Liposomescan be in a range from about 25 nm to about 2000 nm.

In another embodiment, the biodegradable carrier for a synthetic apo A-Imimetic peptide is a polymerosome. “Polymerosomes” are polymer vesiclesformed from di-block or tri-block copolymers with blocks of differingsolubility. Polymerosomes may be formed by methods such as filmrehydration, electro-formation and double emulsion. In some methods, asimilar manufacturing technique can be used as that of a liposome toform polymerosomes. For example, a polymerosome can be a di-blockcopolymer including a block which is hydrophobic, e.g., poly lacticacid, polycaprolactone, n-butyl acrylate, and another block which ishydrophilic, e.g., poly(ethylene glycol), poly(acrylic acid). Apolymerosome can be in a range from between about 25 nm to about 2000nm.

In another embodiment, the biodegradable carrier for a synthetic apo A-Imimetic peptide is a micelle. A “micelle” is an aggregate of surfactantor polymer molecules dispersed in a liquid colloid. Micelles are oftenglobular in shape, but other shapes are possible, including ellipsoids,cylinders, bilayers, and vesicles. The shape of a micelle is controlledlargely by the molecular geometry of its surfactant or polymermolecules, but micelle shape also depends on conditions such astemperature or pH, and the type and concentration of any added salt.

Micelles can be formed from individual block copolymer molecules, eachof which contains a hydrophobic block and a hydrophilic block. Theamphiphilic nature of the block copolymers enables them to self-assembleto form nanosized aggregates of various morphologies in aqueous solutionsuch that the hydrophobic blocks form the core of the micelle, which issurrounded by the hydrophilic blocks, which form the outer shell. Theinner core of the micelle creates a hydrophobic microenvironment formimetic peptide, while the hydrophilic shell provides a stabilizinginterface between the micelle core and an aqueous medium. Examples ofpolymers which can be used to form micelles include, but are not limitedto, polycaprolactone polyethylene oxide blocks, polyethyleneoxide-β-polypropylene oxide-β-polyethylene oxide triblock copolymer andcopolymers which have a polypeptide or polylactic acid core-formingblock and a polyethylene oxide block. A micelle can be in a range frombetween about 10 nm to about 100 nm.

In another embodiment, the biodegradable carrier for a synthetic apo A-Imimetic peptide is a nano or micro-particle. Various methods can beemployed to formulate and infuse or load the particles with a mimeticpeptide. Representative methods include, but are not limited to,water/oil/water (w/o/w) emulsion followed by solvent evaporation, w/o/wemulsion followed by solvent displacement, emulsion polymerization,interfacial polymerization, “salting out,” interfacial polymerization,electrostatic spraying, spray drying, supercritical CO₂ spraying, flashnano-precipitation, electrohydrodynamic atomization, andelectrospraying. In one example, the particles are prepared by awater/oil/water (w₁/o/w₂) double emulsion method. In the w₁ phase, anfirst aqueous phase is dispersed into the oil phase consisting ofpolymer (or other platform) dissolved in organic solvent (e.g.,dichloromethane) and the synthetic apo A-I mimetic peptide (according toembodiments of the invention) using a high-speed homogenizer. Thepolymer should be hydrophobic or amphiphilic but mostly hydrophobic.Examples of polymers include, but are not limited to,poly(L-lactide-co-glycolide) (PLGA), poly(D,L-lactide-co-glycolide)50:50 (PLGA 50:50), poly(L-lactide), poly(D,L-lactide) (PLA),poly(ε-caprolactone), poly(L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone), cross-linked poly(ethylene glycol)(PEG), PLA-PEG co-polymers, poly-ester-amide co-polymers (PEA), andpolyphosphazines. Other examples which may be used as a platform includecollagen, gelatin, fibrin, or alginate. The primary water-in-oil (w/o)emulsion is then dispersed in a second aqueous solution containing apolymeric surfactant, e.g., poly(vinyl alcohol) (PVA) or PEG, andfurther homogenized to produce a w/o/w emulsion. After stirring forseveral hours, the particles are collected by filtration. Amicroparticle can be in a range from about 5 μm to about 200 μm,preferably 10 μm to 50 μm. A nanoparticle can be in a range from betweenabout 10 nm to about 1200 nm, preferably about 80 nm to about 1000 nm.

In one particular embodiment in which an apo A-I mimeticpeptide/phospholipid complex is desired in situ, a non-complexed mimeticpeptide and a phospholipid capable of complexing with the mimeticpeptide may both be encapsulated within a sustained-release carrier.Alternatively, a non-complexed mimetic peptide may be encapsulatedwithin a first sustained-release carrier and a phospholipid capable ofcomplexing with the mimetic peptide may be encapsulated within a seconddifferent sustained-release carrier. It is generally thought that an apoA-1 mimetic peptide complexes with a phospholipid through non-covalentinteractions such as hydrophobic and charge interactions. Theseinteractions can be interrupted by surfactants or solvents which arerequired for some carrier formulation preparations. Thus, in the case ofin situ methods, each component, e.g., a mimetic peptide and aphospholipid, can be separately formulated within different carriers orwithin the same carrier in a non-complexed state so as to avoidpremature separation of the mimetic peptide/phospholipid complex. Forexample, a mimetic peptide, e.g., SEQ ID NO: 4, can be associated with afirst population of sustained-release nanoparticles fabricated by aw/o/w emulsion method and a phospholipid, e.g., DMPC, can be associatedwith a second population of sustained-release nanoparticles fabricatedby a w/o/w emulsion method. The processing conditions and materials mayvary between the w/o/w methods depending on the nature of the moleculebeing encapsulated. The first population can be mixed with the secondpopulation wherein the mimetic peptide remains in a non-complexed statewithin the first population of nanoparticles and the phospholipidremains within the second population until delivery thereof. When thefirst and second populations of nanoparticles are released at atreatment site, e.g., vulnerable plaque (VP) build-up within a bloodvessel, it is anticipated that in situ complexing will occur between themimetic peptide and the phospholipid upon sustained-release thereof.Sustained-release may be controlled by, for example, particle size andmorphology, polymer composition, polymer molecular weight, fabricationtechnique and conditions, or surfactants used in the fabricationthereof. Having control of release rates allows a fine-tuned releaserate for a particular therapy and pharmacokinetic (PK) response.

In another particular embodiment, a first population ofsustained-release particles, e.g., microparticles, can be combined witha second population of sustained-release particles, e.g.,microparticles, wherein the first and second populations differ from oneanother by at least one characteristic including, size, material, orporosity, and, wherein the first and second populations are combined.Each of the populations of nanoparticles may include a mimetic peptide,e.g., SEQ ID NO: 4, a phospholipid capable of complexing with themimetic peptide, e.g., DMPC, or a complex of the mimetic peptide and thephospholipid, wherein the mimetic peptide, the phospholipid, or thecomplex is encapsulated within (or associated with) the particles.Generally, a sustained-release particle undergoes at least two phases oftreatment agent release—the initial burst and the subsequent nearlyzero-order release. In the context of this application, “burst” refersto the amount of treatment agent released in one day or any shortduration divided by the total amount of treatment agent (which isreleased for a much longer duration). “Zero-order release” is the amountof treatment agent released at a constant rate. For any given populationof sustained-release particles, the population has a certain releaseprofile for releasing treatment agent which is based on various factorsincluding, but not limited to, particle size, material, drug loading,drug-material interactions, or porosity of the particles. The releaseprofile may be different depending on whether the distribution ismulti-modal or unimodal. FIG. 1E shows a chart illustrating thepredictive values of treatment agent release from both monodisperse andpolydisperse nanoparticles.

EXAMPLE 3

In a PLGA 50:50 (molecular weight 72.3 Daltons) microparticle releasesystem, the particle size is approximately 1 to 2 μm with 10% loading ofpeptide, i.e., SEQ ID NO.: 4. Certain amounts of microparticles wereincubated in PBS at 37° C., pH 7.4. Periodically, the buffered water wasreplaced and the amount of peptide released was determined by measuringthe concentration of treatment agent in the solution that was removed.FIG. 1F, chart (xiii) illustrates release profiles over days for 1.2 μmaverage size microparticles and 1.8 μm average size microparticles. Theparticle size is controlled by adjusting certain parameters, such as, inthis case, w₁/o/w₂ emulsion evaporation. It has been found that with ahigher molecular weight of the polymer, controlling parameters such asthe evaporation rate, i.e., slower, the amount of surfactant, i.e.,less, and a higher ratio of w₁/w₂, will result in larger particles. FIG.1F, chart (xiv) illustrates a release profiles over days for a 1:1 ratioof 1.2 μm average size microparticles and 1.8 μm average sizemicroparticles. Beneficially, the size distribution can modulate thepenetration efficiency, the permeation gradient, and the overall releaserate of the treatment agent when delivered to a treatment site.

In order to facilitate targeting of the carrier, e.g., a nanoparticle,into a treatment site, e.g., VP build-up within a blood vessel, thecarrier may be coated. In some embodiments, the coating may be specific.A specific moiety may be a targeting moiety specific for the VP, such asan antibody or an antibody fragment such as an anti-intercellularadhesion molecule, an anti-vascular cellular adhesion molecule, ananti-integrin, an anti-platelet endothelial cell adhesion molecule, ananti-thrombomodulin, an anti-E-selectin, an anti-P-selectin, andanti-L-selectin, an anti-fibronectin, an anti-sialyl-Lewis glycan, ananti-endothelial clycocalyx protein, an anti-cadherin, ananti-vitronectin, or a combination thereof. Other examples of specifictargeting moieties include aptamers such as anti-junction adhesionmolecules and anti-leukocyte adhesion molecules. In other embodiments,the coating may be non-specific. A non-specific moiety may be along-chain saturated or unsaturated fatty acid or a ceramide. Thespecific or non-specific moieties may be attached to the surface of thenanoparticle by a bio-conjugation method. For example, a carboxy-PEG maybe incorporated into the surface of the nanoparticle during preparationand then activated by activation of the terminal carboxyl group with,for example, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride or N-hydroxysuccinimide (EDCI/NHS) by methods known in theart of bioconjugation which have been described in, for example,Hermanson, G., et al., Bioconjugation Techniques, Academic Press 1996:The resultant activated carboxyl-NHS polymeric particles (NP) can thenbe reacted with an amino group on the targeting moiety. Alternatively,if the targeting moiety has a carboxy group, the procedure may be toactivate the moiety by methods known by one of ordinary skill in the artand then reacting the resultant activated moiety with an amino-PEG onthe NP.

In an embodiment in which PEG is used (as a platform or surfactant), itis anticipated that PEG will migrate to the surface of the particle toprevent premature release of the synthetic apo A-I mimetic peptideduring therapeutic application, thus achieving sustained-release of themimetic peptide. In another embodiment, the surface of the particle canbe modified with a cardiovascular targeting molecule to achievetargeting and sustained-release of the mimetic peptide. Such targetingmolecules can also be covalently bound to the polymer or other platform,or even to the mimetic peptide itself. An example of a cardiovasculartargeting molecule is CRPPR (SEQ ID NO: 11) peptide. In yet anotherembodiment, a hydrophobic sacrificial layer can be coated onto theparticle to mitigate premature peptide release. An example of a materialwhich may be used for the sacrificial layer is a polyanhydride layermade of CPPP (SEQ ID NO: 12), or, alternatively, bioabsorbable iron- orphosphorous-doped glass or ceramic. It is anticipated that a sacrificiallayer-coated particle having the mimetic peptide will be suitable fororal delivery since such a coating should be able to withstand theenvironment of the gastrointestinal tract.

In another embodiment, the biodegradable carrier for a synthetic apo A-Imimetic peptide is a core-shell particle. Core-shell particles can beformed using various techniques such as, for example, electrospraying.In one exemplary method of fabricating core-shell particles, a firstliquid solution (L₁) may be supplied to an outer tube by a pump and asecond different liquid solution (L₂) may be supplied to an inner tubeby a pump to form the core-shell particles. Solution L₁ may be theprecursor solution that forms the (hydrophobic or hydrophilic) “shell”while solution L₂ may be the precursor solution that forms the(hydrophilic or hydrophobic) “core” of the particles that will beeventually collected on a collection target as the electrospray systemis being operated. By creating core-shell particles encapsulating themimetic peptide, different release profiles may be obtained as the coreand shell independently (or not independently) erode after delivery to atreatment site over a period of time (condition dependent).

In some embodiments, the sustained-release carrier is a microfiber ornanofiber fabricated by electrospinning. “Electrospinning” is a processby which microfibers are formed by using an electric field to draw apolymer solution from the tip of a capillary to a collector. A voltageis applied to the polymer solution which causes a stream of solution tobe drawn toward a grounded collector. Electrospinning generates a web offibers which can be subsequently processed into smaller lengths, i.e.,microfibers or nanofibers, by, for example, cryogenic grinding. In someembodiments, nanofibers may be dispersed in, coated with, or coaxiallyspun with hydrogel materials.

Examples of sustained-release polymers which can be used inelectrospinning include, but are not limited to, PLGA, PLA or PLA-PEEPco-polymers, PEA, polyphosphazines and collagen. In one method, themimetic peptide, or phospholipid, or mimetic peptide/phospholipidcomplex is mixed with a bioerodable polymer solution, a solvent and asurfactant. Examples of surfactants can include, but are not limited to,anionic or cationic surfactants. Useful anionic surfactants include, butare not limited to, bis(2-ethylhexyl) sodium sulfosuccinate (AOT), his(2-ethylhexyl) phosphate (NaDEHP), tauroglycocholate, and sodium laurylsulfate. A useful cationic surfactant is tetradecyltrimethyl-ammoniumbromide (TTAB). Other surfactants can include poloxamers, such asPLURONICS, and polysorbates, such as TWEEN surfactants. Examples ofsolvents include, but are not limited to, hexafluoroisopropanol,acetone, and dichloromethane. The polymer solution is then subjected toelectrospinning. As the solvent evaporates during electrospinning, thetreatment agent incorporates and distributes within the polymer bynon-covalent interactions. The resultant microfibers which can be fromabout 0.05 μm to about 10 μm in diameter form a non-woven web which maythen be processed into smaller lengths of about 0.5 μm to about 100 μm.

In one fabrication embodiment, fibers can be electrospun from collagenand elastin dissolved in hexafluoroisopropanol, forming a polymersolution. A treatment agent can be added to the polymer solution. Asurfactant and a stabilizer can be used to evenly disperse the treatmentagent in the solvent. The polymer solution can then be loaded into asyringe and placed in a syringe pump for metered dispensing at apredetermined rate. A positive output lead of a high voltage supply canbe attached to a needle on the syringe. The needle can be directed to astainless steel grounded target placed approximately 10 cm from theneedle tip, which can be rotated at a predetermined speed to ensure aneven coating. The distance of the needle from the target can be varieddepending upon the diameter of the fibers needed. The resultantmicrofibers are from about 0.05 μm to about 10 μm in diameter and theresulting non-woven mat of fibers can then be processed into smallerlengths of about 0.5 μm to about 100 μm.

Nanofibers may also be formed from self-assembled peptides. Nanorods canbe formed by methods known by those skilled in the art, such as thosedescribed in J. D. Hartgerink, et al., Self-Assembly and Mineralizationof Peptide Amphiphile Nanofibers. Science, 294 (2001):1685-1688; J. D.Hartgerink, et al., Peptide-Amphiphile Nanofibers: A versatile scaffoldfor the preparation of self-assembling materials. PNAS, 99 (2002):5133-5138.

In a further embodiment, the carrier is a gel. A “gel” is an apparentlysolid, jelly-like material formed from a colloidal solution. By weight,gels are mostly liquid, yet they behave like solids. Representatively,the gel is a solution of degradable polymers. For example, the gel canbe an inversion gel of a biodegradable polymer in organic media. Anexample is PLA dissolved in benzyl benzoate containing the mimeticpeptide. In some embodiments, the gel is a biodegradable, viscous gel.For example, the gel can be a solution of sucrose acetate isobutyrate inethanol/water combined with the mimetic peptide. In an example where thegel includes a water-miscible organic solvent plus a polymer, a processof phase inversion occurs when the gel is introduced into the body. Asthe solvent diffuses out, and the water diffuses in, the polymer phaseinverts, or precipitates, forming a depot of varying porosity andmorphology depending on the composition. Gels can also consist of watersoluble polymers in an aqueous carrier. These can provide a fasterrelease of a peptide (e.g., a mimetic peptide), drug or other agent.

In a still further embodiment, the carrier to be used with a syntheticapo A-I mimetic peptide is a lipid-coated microbubble (LCM) includingthe peptide. Peptides can be incorporated into the microbubbles in anumber of different ways, including binding of a peptide to themicrobubble shell and attachment of site-specific ligands.Perfluorocarbon-filled albumin microbubbles avidly bind proteins andsynthetic peptides and are sufficiently stable for circulating in thevasculature as blood pool agents. These microbubbles act as carriers ofthese agents until a site of interest is reached. Ultrasound appliedover the skin surface can then be used to burst the microbubbles at atreatment site, causing localized release of the peptide or protein.Albumin-encapsulated microbubbles have also demonstrated a property toadhere to a vessel wall. These microbubbles provide targeted deliverywithout the application of ultrasound. Microbubbles have also been shownto directly take up genetic material, such as plasmids and adenovirus,and phospholipid-coated microbubbles have a high affinity for certaindrugs.

The mechanisms by which ultrasound facilitates the delivery of drugs andgenes result from an interplay among the therapeutic agent, themicrobubble characteristics, the target tissue, and the nature ofultrasound energy. The presence of microbubbles in the insonified fieldreduces the peak negative pressure needed to enhance delivery withultrasound. This occurs because the microbubbles act as nuclei forcavitation, decreasing the threshold of ultrasound energy necessary tocause this phenomenon. The results of optical and acoustical studieshave suggested the following mechanisms for microbubble destruction byultrasound: gradual diffusion of gas at low acoustic power; formation ofa shell defect with diffusion of gas; immediate expulsion of themicrobubble shell at high acoustic power; and dispersion of themicrobubble into several smaller bubbles.

In an alternative embodiment, the spatially distinct hydrophobic andcharged domains of a synthetic apo A-I mimetic peptide may hold abioactive typically used in cardiovascular treatment. In that sense, themimetic peptide itself acts as the “carrier.” Examples of bioactivesinclude, but are not limited to, statins such as atorvastatin,cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin,pravastatin, rosuvastatin, and simvastatin; nitroglycerin; Acyl CoAcholesterol: acyltransferase (ACAT) inhibitor; anti-inflammatorysteroids such as corticosteroids; singlet oxygen generators such asporphyrins and sub-classes thereof, such as texaphyrins, sapphyrins,pentaphyrins, porphycenes and porphyrin vinylogues in addition to tinethyl etiopurpurin (PURLYTIN), rostaporfin (PHOTREX), verteporfin(VISUDYNE), and TEXAPRIM; phthalocyanines; immunosuppressant oranti-cancer agents such as paclitaxel, sirolimus, everolimus, ABT-578,and eptoposide.

Combination Therapies

Acyl CoA Cholesterol: Acyltransferase Inhibitors

Apo A-I or apo A-I-related molecules (e.g., peptides) have been shown toremove cholesterol from atherosclerotic lesions through reversecholesterol transport. In general, apo A-I has the ability to removefree cholesterol from cell membranes, however, esterified cholesterolmainly located in lipid droplets may not be directly removed by reversecholesterol transport.

Acyl CoA cholesterol: acyltransferase (ACAT) is an enzyme that convertsfree cholesterol into cholesterol esters, and is responsible for thedeposition of cholesterol in cells as cholesterol esters. Inhibition ofthe enzyme can increase the amount of free cholesterol that can beremoved by apo A-I. In addition, oral administration of an ACATinhibitor in rabbits prevents intimal hyperplasia induced by ballooninjury through prevention of foam cell accumulation, and the effectappears independent of plasma cholesterol concentration.

ACAT is a membrane protein and the active center is located inside ofthe membrane. Thus, most ACAT inhibitors are highly hydrophobic and aretherefore inadequate for oral administrations.

In accordance with other embodiments of the invention, a method of localor regional delivery of an ACAT inhibitor is described. The ACATinhibitor may be delivered as a single treatment agent or in combinationwith apo A-I therapy as described in embodiments of the invention. Localadministration of ACAT inhibitors to the blood vessels in combinationwith mimetic peptides according to embodiments of the invention canmaximize reverse cholesterol transport (e.g., reverse cholesterolefflux). This may improve the suppression of lipid accumulation inmacrophage, of macrophage activity, and of smooth muscle cellproliferation. In addition, potential over-production of freecholesterol by inhibition of ACAT, which could cause cell damage, may beminimized by the treatment with mimetic peptides according toembodiments of the invention.

The amount of ACAT inhibitors in regional therapy formulations would besmall and systemic exposure of the ACAT inhibitors would be negligible,thus, inherent side effects of ACAT inhibitors, such as liver andadrenal toxicity can be circumvented. Examples of ACAT inhibitors arelisted as follows, but other ACAT inhibitors may be used as well: CP113,818 (Pfizer), C1-1011 (Pfizer), (Avasimibe), CI 976, CL-277,082,Eflucimibe (bioMrieux-Pierre Fabre/Eli Lilly), CS-505 (SankyoPharma/Kyoto Pharma), (Pactimibe), KY-455 (Kyoto Pharma), F-1394(Fujirebio Inc.), F 12511, NTE-122 (Nissin Food Products Co., Ltd.), PD140296 (Parke-Davis), PD 128042 (Parke-Davis), PD 132301-2(Parke-Davis), (Octimibate), DuP 128, DuP 129, 58-035 (Sandoz), HL-004,SMP-500 (Sumitomo Pharma), SMP-797, SM-32504 (Sumitomo Pharma),SKF-99085 (Glaxo Smith-Kline), E5324, R-755 (Nihon Nohyaku), FR145237(Fujisawa Pharmaceutical Co., Ltd.), FR129169 (Fujisawa PharmaceuticalCo., Ltd.), FR186054, YM-17E (Yamanouchi Pharma), YM750 (YamanouchiPharma), Tamoxifen, MCC-147, YIC-708-424 (Yakult), TS-962 (Taisho),K-604 (Kowa), FCE-28654A (Pharmacia & Upjohn Inc.), CEB-925 (Wyeth).

In one embodiment, an apo A-I synthetic peptide is part of a deliverycomposition. In addition to the peptide, the composition may include aphospholipid such as DMPC and a buffer such as phosphate buffered saline(PBS) that may serve to maintain an osmotic pressure and control the pHof the delivery composition. As noted above, the phospholipid componentis optional, particularly with peptides that have demonstrated anability to function in a non-complex form, such as the 18-mer L4-F andD4-F.

Other

In some embodiments, if the bioactive is a hydrophobic molecule, thebioactive may facilitate self-assembly of the mimetic peptide by servingas a nidus for self-assembly. Examples of hydrophobic molecules include,but are not limited to, hydrophobic pro-drugs such as lipid-conjugatedsmall molecule agents. In one embodiment, a hydrophobic vasodilatoryagent such as isosorbide-5-mononitrate can be conjugated through itshydroxyl group to a fatty acid to form a pro-drug ester that can slowlybe released upon hydrolysis of the ester bond.

In some embodiments, it is anticipated that a synergistic effect can beachieved by combination delivery or co-delivery of a peptide mimetic andanother agent. For example, a peptide mimetic may be delivered(simultaneously or subsequently) with cholesterol esterase. Thecholesterol esterase converts esterified cholesterol to free cholesterolthereby facilitating reverse cholesterol transport by the mimeticpeptide. In another example, a peptide mimetic may be delivered(simultaneously or subsequently) with a nitric oxide-releasing agentsuch as PEA-TEMPO or poly(L-arginine).

Delivery Methods

In one embodiment, a method includes advancing a delivery device througha lumen of a blood vessel to a particular region in a blood vessel suchas a lesion area in a coronary atherosclerotic region. A compositionincluding a carrier and an apo A-I synthetic peptide is then introducedinto a wall of the blood vessel at the lesion area or in theperi-vascular site. By introducing the apo A-I synthetic peptide intothe lesion area, significantly less peptide may be used relative to theamount that might be used in a systemic delivery treatment regimen. Inone embodiment, an amount target of a five microgram apo A-Ipeptide/kilogram (μg/kg) for an adult human is suitable. The deliverydevice may also be used to introduce an ACAT inhibitor(s), possibly insimilar amounts, into or beyond the blood vessel at or adjacent thelesion area.

Referring now to the drawings, FIG. 1 illustrates one embodiment of adelivery apparatus. In general, the delivery apparatus provides a systemfor delivering an apo A-I synthetic peptide or a treatment compositionincluding an apo A-I synthetic peptide, to or through a desired area ofa blood vessel (a physiological lumen) or tissue in order to treat alocalized area of the blood vessel. The delivery apparatus is similar incertain respects to the delivery apparatus described in commonly-owned,U.S. patent application Ser. No. 09/746,498 (filed Dec. 21, 2000),titled “Local Drug Delivery Catheter with Retractable Needle,” of Chowet al. (issued as U.S. Pat. No. 6,692,466) and U.S. patent applicationSer. No. 10/749,354 (filed Dec. 31, 2003), titled “Modified NeedleCatheter for Directional Orientation Delivery” of Chan, et al. Each ofthese applications is incorporated herein by reference. The deliveryapparatus described is suitable, in one embodiment, for a percutaneousdelivery of a treatment agent where a desired form of the treatmentagent is introduced through a single catheter needle.

Referring to FIG. 1, the delivery apparatus includes catheter assembly100, which is intended to broadly include any medical device designedfor insertion into a blood vessel or physiological lumen to permitinjection and/or withdrawal of fluids, to maintain the patency of thelumen, or for any other purpose. In one embodiment, catheter assembly100 is defined by elongated catheter body (cannula) 112 having proximalportion 113 and distal portion 114. In one embodiment, proximal portion113 may reside outside a patient during a procedure while distal portion114 is placed at a treatment site, for example, within coronary bloodvessel 117.

Catheter assembly 100 includes catheter body 112 having a lumentherethrough extending from proximal portion 113 to distal portion 114.In this example, guidewire cannula 116 is formed within catheter body112 for allowing catheter assembly 100 to be fed and maneuvered over aguidewire (guidewire 118 shown at this point within a lumen of guidewirecannula 116). Guidewire cannula 116 may extend from proximal portion 113to distal portion 114, thus describing an over the wire (OTW) assembly.In another embodiment, typically described as a rapid exchange (RX) typecatheter assembly, guidewire cannula 116 extends only through a portionof catheter body 112, for example, beginning and ending within distalportion 114. An RX type catheter assembly is shown. It is appreciatedthat guidewire 118 may be retracted or removed once catheter assembly100 is placed at a region of interest, for example, within a bloodvessel (e.g., artery or vein).

In the embodiment of FIG. 1, catheter assembly 100 includes balloon 120incorporated at distal portion 114 of catheter assembly 100. Balloon 120is an expandable body in fluid communication with inflation cannula 128disposed within catheter body 112. Inflation cannula 128 extends fromballoon 120 within distal portion 114 through inflation port 148 atproximal portion 113 (e.g., at a proximal end of catheter assembly 100).Inflation cannula 128 is used to deliver a fluid to inflate balloon 120.

In the embodiment shown in FIG. 1, balloon 120 is in an expanded orinflated state that occludes blood vessel 117. Balloon 120 isselectively inflatable to dilate from a collapsed configuration to adesired or controlled expanded configuration. Balloon 120 can beselectively inflated by supplying a fluid (e.g., liquid) into a lumen ofinflation cannula 128 at a predetermined rate of pressure throughinflation port 148. Likewise, balloon 120 is selectively deflatable toreturn to a collapsed configuration or deflated profile.

In one embodiment, balloon 120 can be defined by three portions: distaltaper wall 126, medial working length 124, and proximal taper wall 122.In one embodiment, proximal taper wall 122 can taper at any suitableangle θ, typically between about 15° to less than about 90°, whenballoon 120 is in an expanded (inflated) configuration.

Balloon 120 can be made from any suitable material, including, but notlimited to, polymers and copolymers of polyolefins, polyamides,polyester and the like. The specific material employed should becompatible with inflation or expansion fluid and must be able totolerate the pressures that are developed within balloon 120. Onesuitable material is an elastomeric nylon such as PEBAX™, a condensationpolymerized polyether block polyamide. PEBAX™ is a trademark of ATOCHEMCorporation of Puteaux, France. Other suitable materials for balloon 120include, but are not limited to, a biocompatible blend of polyurethaneand silicone, or a styrenic block copolymer (SBC) or blend of SBCs.Distal taper wall 126, medial working length 124, and proximal taperwall 122 can be bound together by seams or be made out of a singleseamless material. A wall of balloon 120 (e.g., at any of distal taperwall 126, medial working length 124 and/or proximal taper wall 122) canhave any suitable thickness so long as the thickness does not compromiseproperties that are critical for achieving optimum performance. Relevantproperties include, but are not limited to, high burst strength, lowcompliance, good flexibility, high resistance to fatigue, the ability tofold, the ability to cross and recross a desired region of interest oran occluded region in a physiological lumen and low susceptibility todefects caused by handling. By way of example, not limitation, asuitable thickness of a balloon wall can be in the range of about 0.0005inches to 0.002 inches, the specific specifications depending on theprocedure for which balloon 120 is to be used and the anatomy and sizeof the target lumen in which balloon 120 is to be inserted.

Balloon 120 may be inflated by the introduction of a fluid (e.g.,liquid) into inflation cannula 128 (through inflation port 148 at apoint outside a physiological lumen). Liquids containing therapeuticand/or diagnostic agents may be used to inflate balloon 120. In oneembodiment, balloon 120 may be made of a material that is permeable tosuch therapeutic and/or diagnostic agents thus providing a method ofdelivering a therapeutic and/or diagnostic agent at a treatment site inaddition to an apo A-I peptide, a treatment composition including an apoA-I peptide or an ACAT inhibitor. To inflate balloon 120, a suitablefluid may be supplied into inflation cannula 128 at a predeterminedpressure, for example, between about one and 20 atmospheres (atm). Aspecific pressure depends on various factors, such as the thickness ofthe balloon wall, the material of which balloon 120 is made, the type ofsubstance employed, and the flow rate that is desired.

Catheter assembly 100, in the embodiment shown in FIG. 1 also includesdelivery cannula 130 and delivery cannula 132 each connected to proximaltaper wall 122 of balloon 120 and extending at a proximal end, in oneembodiment, into a portion of catheter body 112 of catheter assembly100. Representatively, a suitable length for delivery cannula 130 anddelivery cannula 132 is on the order of three to 6.5 centimeters (cm).Delivery cannula 130 and delivery cannula 132 can be made from anysuitable material, such as polymers and copolymers of polyamides,polyolefins, polyurethanes, and the like. Catheter assembly 100, in thisview, also includes needle 134 and needle 136. Needle 134 and needle 136extend from distal portion 114 to proximal portion 113 of catheterassembly 100. At distal portion 114, needle 134 is slidably disposedthrough a lumen of delivery cannula 130 and needle 136 is slidablydisposed through a lumen of delivery cannula 132. Thus, a dimension ofdelivery cannula 130 and delivery cannula 132 are each selected to besuch to allow a delivery device such as a needle to be movedtherethrough. Representatively, delivery cannula 130 has an innerdiameter (lumen diameter) on the order of 0.002 inches to 0.020 inches(e.g., 0.0155 inches) and an outer diameter on the order of 0.006 inchesto 0.05 inches (e.g., 0.0255 inches). FIG. 1 shows catheter assembly 100with each of needle 134 and needle 136 deployed in an extendedconfiguration, i.e., extending from an end of delivery cannula 130 anddelivery cannula 132, respectively. In a retracted configuration, theneedles retract proximally into the delivery cannula lumens. Althoughtwo needles are shown, in another embodiment, catheter assembly mayinclude only a single needle (and single delivery cannula) or mayinclude more than two needles (and more than two delivery cannulas).

FIG. 1 shows delivery cannula 130 and delivery cannula 132 eachconnected to an exterior surface of balloon 120. Specifically, a distalend of each of delivery cannula 130 and delivery cannula 132 extend to apoint equivalent to or less than a length of proximal taper wall 122 ofballoon 120. One suitable technique for connecting delivery cannula 130or delivery cannula 132 to balloon 120 is through an adhesive. Asuitable adhesive includes a cyanoacrylate (e.g., LOCTITE 414™)adhesive, particularly where the balloon material is a PEBAX™ material.

Catheter assembly 100 in the embodiment shown in FIG. 1 also includessheath ring 125. Sheath ring 125 is positioned over, in this embodiment,guidewire cannula 116, inflation cannula 128, delivery cannula 130, anddelivery cannula 132, respectively. In one embodiment, sheath ring 125functions to inhibit delamination of the delivery cannulas from proximaltaper wall 122 of balloon 120 and, where thermally sealed to the variouscannulas may reduce the spacing (on a proximal side of sheath ring 125)of the cannulas. Thus, a distal end of sheath ring 125 is placed, in oneembodiment, at a point immediately proximal to where a delivery cannulawill rotate, bend or plicate in response to the expansion or inflationof balloon 120. In one embodiment, sheath ring 125 is a biocompatiblematerial that is capable of connecting to (e.g., bonding to) a materialfor balloon 120 and to a material for each of the noted cannulas that itsurrounds. Representatively, a body of sheath ring 125 has a length froma proximal end to a distal end on the order of 0.25 millimeters (mm) to0.75 mm, such as 0.5 mm.

As noted above, each delivery cannula (e.g., delivery cannula 130,delivery cannula 132) plicates or bends distal to sheath ring 125 withthe inflation of balloon 120. Thus, the path to be traveled by eachneedle (e.g., needle 134 and needle 136) includes this bend orplication. To facilitate a travel through a bend or plication region ineach delivery cannula and to inhibit puncturing of the respectivedelivery cannula, each delivery cannula may include a deflector disposedalong an interior wall. Representatively, a suitable deflector includesa ribbon of thin, generally flexible and generally resilient material(e.g., thickness on the order of about 0.0005 inches to about 0.003inches and width on the order of about 0.005 inches and 0.015 inches).Suitable deflector materials, dimensions and connections within acatheter assembly are described in commonly-owned, U.S. patentapplication Ser. No. 09/746,498 (filed Dec. 21, 2000), titled “LocalDrug Delivery Catheter with Retractable Needle,” of Chow et al. (issuedas U.S. Pat. No. 6,692,466) and U.S. patent application Ser. No.10/749,354 (filed Dec. 31, 2003), titled “Modified Needle Catheter forDirectional Orientation Delivery.” of Chan, et al.

Referring again to FIG. 1, proximal portion 113 of catheter assembly 100is intended, in one embodiment, to reside outside a patient while theremainder of catheter assembly 100 is percutaneously introduced into,for example, the cardiovascular system of a patient via a brachial, aradial or a femoral artery. In this embodiment, proximal portion 113 ofcatheter assembly 100 includes hub 140. Hub 140 includes needle 134 andneedle 136, and inflation cannula 128. In one embodiment, relative tothe materials for the various cannulas described, a housing of hub 140is a hard or rigid polymer material, e.g., a polycarbonate oracrylonitrile butadiene styrene (ABS). A distal end of hub 140 has anopening to accommodate a proximal end of catheter body 112. Hub 140 alsohas a number of cavities at least partially therethrough (extending in adistal to proximal direction) to accommodate needle 134 and needle 136,and inflation cannula 128. A proximal portion of hub 140 flares toseparate a spacing between the needles, and inflation cannula 128.

FIG. 1 shows a proximal end of needle 134 and needle 136 each connected(e.g., through an adhesive) to respective injection port 144 andinjection port 146. In one embodiment, each injection port includes aluer fitting for conventional syringe attachment. Each injection portallows for the introduction of treatment agent 150, including but notlimited to an apo A-I peptide, a treatment agent including an apo A-Ipeptide and/or an ACAT inhibitor. It is appreciated that treatment agent150 introduced at injection portion 144 and injection port 146 may bethe same or different (e.g., a treatment agent including an apo A-Ipeptide versus an ACAT inhibitor, a drug, or other cellular component).In this embodiment, inflation cannula 128 terminates at the distal endof balloon inflation port 148.

In one embodiment, catheter assembly 100 also includes or can beconfigured to include an imaging assembly. Suitable imaging assembliesinclude ultrasonic imaging assemblies, optical imaging assemblies, suchas an optical coherence tomography (OCT) assembly, magnetic resonanceimaging (MRI). One embodiment of catheter assembly 100 illustrated inFIG. 1 may include an OCT imaging assembly.

OCT uses short coherent length light (typically with a coherent lengthof about 10 to 100 microns) to illuminate the object (e.g., blood vesselor blood vessel walls). Light reflected from a region of interest withinthe object is combined with a coherent reference beam. Interferenceoccurs between the two beams only when the reference beam and reflectivebeam have traveled the same distance. One suitable OCT setup may besimilar to ones disclosed in U.S. Pat. Nos. 5,465,147; 5,459,570;5,321,501; 5,291,267; 5,365,125; and 5,202,745. A suitable opticalassembly for use in conjunction with a catheter assembly is made withfiber optic components that, in one embodiment, can be passed throughthe guidewire lumen (e.g., guidewire cannula 116 of FIG. 1).

The catheter assembly described with reference to FIG. 1 may be used tointroduce an apo A-I peptide, a treatment composition including an apoA-I peptide and/or an ACAT inhibitor such as described above at adesired location. FIG. 2 illustrates one technique. FIG. 3 presents ablock diagram of one technique. With reference to FIGS. 2 and 3 andcatheter assembly 100 of FIG. 1, in a one procedure, guidewire 118 isintroduced into, for example, an arterial system of a patient (e.g.,through the femoral artery) until the distal end of guidewire 118 isupstream of a narrowed lumen of the blood vessel (e.g., upstream oflesion area 285 in vessel 270). Catheter assembly 100 is mounted on theproximal end of guidewire 118 and advanced over the guidewire 118 untilcatheter assembly 100 is position as desired. In the example shown inFIG. 2, catheter assembly 100 is positioned so that a medial workinglength of balloon 120 and delivery cannula 130 are at or adjacent thenarrowed lumen of vessel 270 (block 310). Imaging techniques may be usedto place catheter assembly 100. Once balloon 120 is placed and subjectto low inflation pressure, guidewire 118 is removed and replaced in oneembodiment with an optical fiber for imaging (e.g., OCT) (block 320).

In this example, vessel 270 is viewed and the lesion area is identifiedor a thickness of the atherosclerotic lesion is imaged (and possiblymeasured) (block 320). At this point, balloon 120 is dilated as shown inFIG. 1 by, for example, delivering a fluid to balloon 120 throughinflation cannula 128. The inflation of balloon 120 causes deliverycannula 130 to move proximate to or contact the blood vessel wall at thelesion area. Needle 136 is then advanced a distance into the lesion(e.g., atherosclerotic lesion) (block 340). A real time image may beused to advance needle 136. Alternatively, the advancement may be basedon a measurement of the blood vessel wall or lesion boundary derivedfrom an optical image. Once in position, an apo A-I peptide or atreatment composition including an apo A-I peptide is introduced throughneedle 136 to the lesion (block 350).

In an embodiment where an ACAT inhibitor is also introduced throughcatheter assembly 100, an ACAT inhibitor may be introduced throughneedle 136. In one embodiment, needle 136 may be introduced into a wallof vessel 270 at the lesion area or beyond the vessel (e.g., to aperiadventitial space). An ACAT inhibitor may then be introduced throughinjection port 146.

In the above embodiment, an apo A-I peptide, treatment compositionincluding an apo A-I peptide, and/or ACAT inhibitor is introduceddirectly into a blood vessel wall (e.g., a lesion area of a blood vesselwall). Such introduction may follow (either immediately or at some timethereafter) a percutaneous angioplasty (PTCA) by an expanding balloon.Such introduction may also precede or follow the placement of a stentadjacent a lesion area. FIG. 4 shows a coronary blood vessel (e.g., LCX270) having stent 410 placed adjacent lesion area 285. In oneembodiment, stent 410 may be a drug- or other treatment agent-elutingstent. For example, stent 410 may be coated with an ACAT inhibitor topermit a combination therapy of apo A-I and an ACAT inhibitor. In thisembodiment, following a placement of stent 410, needle 136 may beadvanced through openings in a cage-like structure of stent 410 intolesion area 285 for delivery of an apo A-I peptide or a treatmentcomposition including an apo A-I peptide into lesion area 285.

In the example where stent 410 is a coated stent for eluting an ACATinhibitor, the stent may be composed of a metal, an alloy, a polymer, ora combination thereof and a treatment agent included in a stent coatingor in the body of the stent. Examples of materials used to form stentsinclude, but are not limited to, ELATINITE®, Nitinol (nickel-titaniumalloy), stainless steel, tantalum, tantalum-based alloys, platinum,platinum-based alloys, and other metals and their alloys. Alternatively,stent 410 is composed of a bioabsorbable polymer or biostable polymer. Apolymer or coating is “bioabsorbable” or “biodegradable” when it iscapable of being completely or substantially degraded or eroded whenexposed to either an in vivo environment or an in vitro environmenthaving physical, chemical, or biological characteristics substantiallysimilar to those of the in vivo environment within a mammal. A polymeror coating is “degradable or erodible” when it can be gradually brokendown, resorbed, absorbed and eliminated by, for example, hydrolysis,enzymolysis, metabolic processes, bulk or surface erosion, and the likewithin a mammal. It is to be appreciated that traces of residue ofpolymer may remain following biodegradation. A “biostable” polymer is apolymer that is not bioabsorbable.

Suitable polymers used in embodiments of a material for a body stent 410(i.e., the structural aspect of the stent as opposed to a coating),include, but are not limited to, hydrophobic, hydrophilic, amphiphilic,biodegradable, or a combination thereof. Examples of hydrophobicpolymers include, but are not limited to, poly(ester amide),polystyrene-polyisobutylene-polystyrene block copolymer (SIS),polystyrene, and polyisobutylene. Examples of hydrophilic polymersinclude, but are not limited to, polymers and co-polymers ofhydroxyethyl methacrylate (HEMA); poly(methyl methacrylate) (PMMA); andpoly(ethylene glycol)acrylate (PEGA). Examples of biodegradable polymersinclude, but are not limited to, polymers having repeating units suchas, for example, an α-hydroxycarboxylic acid, a cyclic diester of anα-hydroxycarboxylic, a dioxanone, a lactone, a cyclic carbonate, acyclic oxalate, an epoxide, a glycol, an anhydride, a lactic acid, aglycolic acid, a glycolic acid, a lactide, a glycolide, an ethyleneoxide, an ethylene glycol, or combinations thereof. In some embodiments,the biodegradable polymers include, but are not limited, to polyesters,polyhydroxyalkanoates (PHAs), poly(ester amides), amino acids, PEGand/or alcohol groups, polycaprolactones, poly(L-lactide),poly(D,L-lactide, poly(D,L-lactide-co-PEG) block copolymers,poly(D,L-lactide-co-trimethylene carbonate), polyglycolides,poly(lactide-co-glycolide), polydioxanones, polyorthoesters,polyanhydrides, poly(glycolic acid-co-trimethylene carbonate),polyphosphoesters, polyphosphoester urethanes, poly(amino acids),polycyanoacrylates, poly(trimethylene carbonate), poly(imino carbonate),polycarbonates, polyurethanes, co-poly (ether-esters) (e.g., PEO/PLA),polyakylene oxalates, polyphosphazenes, PHA-PEG, and any derivatives,analogs, homologues, salts, copolymers and combinations thereof.

A composition including an ACAT inhibitor may be included in a stentcoating on stent 410 or included in the body of stent 410 such as, forexample, a biodegradable polymeric stent. The release profile of, forexample, ACAT inhibitor and polymer can be controlled by tailoring thechemical composition and crystallinity of the polymer as the coating orthe bioabsorbable stent material (e.g., the more crystalline, the slowerthe release rate).

In the embodiments described with reference to FIGS. 1-4, a catheterassembly for introducing an apo A-I peptides, a treatment compositionincluding an apo A-I peptide, and/or an ACAT inhibitor into a wall of ablood vessel (e.g., into a lesion area of the blood vessel) isdescribed. In another embodiment, it may be desired to introduce an apoA-I peptide, a treatment composition including an apo A-I peptide and/oran ACAT inhibitor within a blood vessel (i.e., an intra-coronaryintroduction). Such technique may be used, for example, to deliver anapo A-I synthetic peptide including an amino acid sequence in an orderreverse to an order of an endogenous apo A-I peptide and/or an apo A-Ipeptide that is a chimera of helix 1 and helix 9 of apo A-I, optionallywith an amino acid sequence in reverse order. An intra-coronaryintroduction of the apo A-I peptide or a treatment composition includingan apo A-I peptide will promote reverse cholesterol transport fromwithin a lumen of a coronary vessel. Alternatively, an apo A-I peptideor a treatment composition including an apo A-I peptide may beintroduced regionally, such as injected into an accessible arterythrough a needle injection.

FIG. 5 shows blood vessel 517 having catheter assembly 500 disposedtherein. Catheter assembly 500 includes proximal portion 513 and distalportion 514. Proximal portion 513 may be external to blood vessel 517and to the patient. Representatively, catheter assembly 500 may beinserted through a femoral artery and through, for example, a guidecatheter and with the aid of a guidewire to a location in thevasculature of a patient. That location may be, for example, a coronaryartery. FIG. 5 shows distal portion 514 of catheter assembly 500positioned at a treatment site within a coronary blood vessel (bloodvessel 517).

In one embodiment, catheter assembly 500 includes primary cannula 512having a length that extends from proximal portion 513 (e.g., locatedexternal to a patient during a procedure) to connect to the proximal endor skirt of balloon 520. Primary cannula 512 has a lumen therethroughthat includes inflation cannula 528 and delivery cannula 530. Each ofthe inflation cannula 528 and delivery cannula 530 extend from proximalportion 513 of catheter assembly 500 to distal portion 514. Inflationcannula 528 has a distal end that terminates in balloon 520. Deliverycannula 530 extends through balloon 520 (i.e., beyond a distal end orskirt of balloon 520). In another embodiment, catheter assembly 500 doesnot include a balloon or inflation cannula.

Catheter assembly 500 also includes guidewire cannula 516 extending, inthis embodiment, through balloon 520 to a distal end of catheterassembly 500. Guidewire cannula 516 has a lumen sized to accommodate aguidewire (not shown). Catheter assembly 500 may be an over-the-wire(OTW) configuration where guidewire cannula 516 extends from a proximalend (external to a patient during a procedure) to a distal end ofcatheter assembly 500. In another embodiment, catheter assembly 500 is arapid exchange (RX) type catheter assembly where only a portion ofcatheter assembly 500 (a distal portion including balloon 520) isadvanced over the guidewire. FIG. 5 shows an OTW type catheter assembly.

In one embodiment, catheter assembly is introduced into blood vessel 517in a direction of blood flow, such as through a femoral artery to alocation within a coronary artery. Once introduced, balloon 520 isinflated (e.g., with a suitable liquid through inflation cannula 528) toocclude a blood vessel. Following occlusion, an apo A-I peptide or atreatment composition including an apo A-I peptide is introduced throughdelivery cannula 530. FIG. 5 shows treatment agent 550 that may beconnected to delivery port 544 and introduced into delivery cannula 530.As noted above, in one embodiment, the delivery of treatment agent 550will be with the flow of blood through the blood vessel. To retain atreatment agent (e.g., apo A-I) within a location in a blood vessel forat least a minimum period of time, it may be desirable to inflate afirst balloon distal to an injury site (e.g., lesion area) and inflate asecond balloon proximal to the injury site, thus isolating the injurysite between the two inflated balloons. The distal balloon may be partof catheter assembly 500 (e.g., a dual balloon catheter) or a part ofthe guidewire (e.g., a PERCUSURG™ catheter assembly, commerciallyavailable from Medtronic, Inc. of Minneapolis, Minn.)

In an effort to improve the target area of an apo A-I mimetic peptide toa treatment site, such as treatment site 285 in FIG. 2, the treatmentsite may be isolated prior to delivery. FIG. 6 shows an embodiment of acatheter assembly having two balloons where one balloon is locatedproximal to treatment site 285 and a second balloon is located distal totreatment site 285. A stent may optionally be placed adjacent totreatment site. FIG. 6 shows stent 605 that may be a drug-eluting stentcoated with, for example, an ACAT inhibitor. FIG. 6 shows catheterassembly 600 disposed within blood vessel 100. Catheter assembly 600 hasa tandem balloon configuration including proximal balloon 625 and distalballoon 635 aligned in series at a distal portion of the catheterassembly. Catheter assembly 600 also includes primary cannula 615 havinga length that extends from a proximal end of catheter assembly 600(e.g., located external to a patient during a procedure) to connect witha proximal end or skirt of balloon 625. Primary cannula 615 has a lumentherethrough that includes inflation cannula 630 and inflation cannula650. Inflation cannula 630 extends from a proximal end of catheterassembly 600 to a point within balloon 625. Inflation cannula 630 has alumen therethrough allowing balloon 625 to be inflated through inflationcannula 630. In this embodiment, balloon 625 is inflated through aninflation lumen separate from the inflation lumen that inflates balloon635. Inflation cannula 650 has a lumen therethrough allowing fluid to beintroduced in the balloon 635 to inflate the balloon. In this manner,balloon 625 and balloon 635 may be separately inflated. Each ofinflation cannula 630 and inflation cannula 650 extends from, in oneembodiment, the proximal end of catheter assembly 600 through a pointwithin balloon 625 and balloon 635, respectively.

Catheter assembly 600 also includes guidewire cannula 620 extending, inthis embodiment, through each of balloon 625 and balloon 635 through adistal end of catheter assembly. Guidewire cannula 620 has a lumentherethrough sized to accommodate a guidewire. No guidewire is shownwithin guidewire cannula 620. Catheter assembly 600 may be an over thewire (OTW) configuration or a rapid exchange (RX) type catheterassembly. FIG. 6 illustrates an RX type catheter assembly.

Catheter assembly 600 also includes delivery cannula 640. In thisembodiment, delivery cannula 640 extends from a proximal end of catheterassembly 600 through a location between balloon 625 and balloon 635.Secondary cannula 645 extends between balloon 625 and balloon 635. Aproximal portion or skirt of balloon 635 connects to a distal end ofsecondary cannula 645. A distal end or skirt of balloon 625 is connectedto a proximal end of secondary cannula 645. Delivery cannula 640terminates at opening 660 through secondary cannula 645. In this manner,a treatment agent such as apo A-I mimetic peptide may be introducedbetween balloon 625 and balloon 635 positioned between treatment site285.

FIG. 6 shows balloon 625 and balloon 635 each inflated to occlude alumen of blood vessel 100 and isolate treatment site 285. In oneembodiment, each of balloon 625 and balloon 635 are inflated to a pointsufficient to occlude blood vessel 100 prior to the introduction of atreatment agent. A treatment agent, such as apo A-I mimetic peptide isthen introduced through opening 660.

In the above embodiment, separate balloons having separate inflationlumens are described. It is appreciated, however, that a singleinflation lumen may be used to inflate each of balloon 625 and balloon635. Alternatively, in another embodiment, balloon 635 may be aguidewire balloon configuration such as a PERCUSURG™ catheter assemblywhere catheter assembly 600 including only balloon 625 is inserted overa guidewire including balloon 635.

FIG. 7 shows another embodiment of a catheter assembly. Catheterassembly 700, in this embodiment, includes a porous balloon throughwhich a treatment agent, such as apo A-I mimetic peptide, may beintroduced. FIG. 7 shows catheter assembly 700 disposed within bloodvessel 100. Catheter assembly 700 has a porous balloon configurationpositioned at treatment site 285. Catheter assembly 700 includes primarycannula 715 having a length that extends from a proximal end of catheterassembly 700 (e.g., located external to a patient during a procedure) toconnect with a proximal end or skirt of balloon 725. Primary cannula 715has a lumen therethrough that includes inflation cannula 730. Inflationcannula 730 extends from a proximal end of catheter assembly 700 to apoint within balloon 725. Inflation cannula 730 has a lumen therethroughallowing balloon 725 to be inflated through inflation cannula 730.

Catheter assembly 700 also includes guidewire cannula 720 extending, inthis embodiment, through balloon 725. Guidewire cannula 720 has a lumentherethrough sized to accommodate a guidewire. No guidewire is shownwithin guidewire cannula 720. Catheter assembly 700 may be anover-the-wire (OTW) configuration or rapid exchange (RX) type catheterassembly. FIG. 7 illustrates an OTW type catheter assembly.

Catheter assembly 700 also includes delivery cannula 740. In thisembodiment, delivery cannula 740 extends from a proximal end of catheterassembly 700 to proximal end or skirt of balloon 725. Balloon 725 is adouble layer balloon. Balloon 725 includes inner layer 7250 that is anon-porous material, such as PEBAX, Nylon or PET. Balloon 725 alsoincludes outer layer 7255. Outer layer 7255 is a porous material, suchas extended polytetrafluoroethylene (ePTFE). In one embodiment, deliverycannula 740 is connected to between inner layer 7250 and outer layer7255 so that a treatment agent can be introduced between the layers andpermeate through pores in balloon 725 into a lumen of blood vessel 100.

As illustrated in FIG. 7, in one embodiment, catheter assembly 700 isinserted into blood vessel 100 so that balloon 725 is aligned withtreatment site 285. Blood vessel 100 may include stent 710 disposedadjacent treatment site 285. Following alignment of balloon 725 ofcatheter assembly 700, balloon 725 may be inflated by introducing aninflation medium (e.g., liquid through inflation cannula 730). In oneembodiment, balloon 725 is only partially inflated or has an inflateddiameter less than an inner diameter of blood vessel 100 at treatmentsite 285. In this manner, balloon 725 does not contact or only minimallycontacts the blood vessel wall. A suitable expanded diameter of balloon725 is on the order of 2.0 to 5.0 mm for coronary vessels. It isappreciated that the expanded diameter may be different for peripheralvasculature. Following the expansion of balloon 725, a treatment agent,such as apo A-I mimetic peptide is introduced into delivery cannula 740.The treatment agent flows through delivery cannula 740 into a volumebetween inner layer 7250 and outer layer 7255 of balloon 725. At arelatively low pressure (e.g., on the order of two to four atmospheres(atm)), the treatment agent then permeates through the porous of outerlayer 7255 into blood vessel 100.

FIG. 8 shows another embodiment of a catheter assembly suitable forintroducing a treatment agent into a blood vessel. FIG. 8 shows catheterassembly 800 disposed within blood vessel 100. Blood vessel 100 may alsoinclude stent 810 disposed adjacent treatment site 285. Catheterassembly 800 includes primary cannula 815 having a length that extendsfrom a proximal end of catheter assembly 800 (e.g., located external toa patient during a procedure) to connect with a proximal and/or skirt ofballoon 825. Balloon 825, in this embodiment, is located at a positionaligned with treatment site 285 in blood vessel 100.

Disposed within primary cannula 815 is guidewire cannula 820 andinflation cannula 830. Guidewire cannula 820 extends from a proximal endof catheter assembly 800 through balloon 825. A distal end or skirt ofballoon 825 is connected to a distal portion of guidewire cannula 820.

Inflation cannula 830 extends from a proximal end of catheter assembly800 to a point within balloon 825. In one embodiment, balloon 825 ismade of a porous material such as ePTFE. A suitable pore size for anePTFE balloon material is on the order of one μm to 60 μm. The porosityof ePTFE material can be controlled to accommodate a treatment agentflow rate or particle size by changing a microstructure of an ePTFE tapeused to form a balloon, for example, by wrapping around a mandrel.Alternatively, pore size may be controlled by controlling the compactionprocess of the balloon, or by creating pores (e.g., micropores) using alaser.

ePTFE as a balloon material is a relatively soft material and tends tobe more flexible and conformable with tortuous coronary vessels thanconventional balloons. ePTFE also does not need to be folded which willlower its profile and allow for smooth deliverability to distal lesionsand the ability to provide therapy to targeted or regional sites postangioplasty and/or stent deployment.

A size of balloon 825 can also vary. A suitable balloon diameter is, forexample, in the range of two to five mm. A balloon length may be on theorder of eight to 60 mm. A suitable balloon profile range is, forexample, approximately 0.030 inches to 0.040 inches.

In one embodiment, a porous balloon may be masked in certain areas alongits working length to enable more targeted delivery of a treatmentagent. In another embodiment, a sheath may be advanced over a porousballoon (or the balloon withdrawn into a sheath) to allow tailoring of atreatment agent distribution. In another embodiment, a sheath may have awindow for targeting delivery of the treatment agent through a porousballoon. In another embodiment, a liner inside a porous balloon may beused to target preferential treatment agent delivery. For example, theliner may have a window through which a treatment agent is delivered,e.g., on one side of a liner for delivery to one side of a vessel wall.This type of configuration may be used to address eccentric lesions.

In an alternative embodiment, rather than using a porous material likeePTFE for forming a porous balloon (e.g., balloon 825 in FIG. 8), aconventional balloon material such as PEBAX, Nylon or PET may be usedthat has tens or hundreds of micropores around its circumference fortreatment agent diffusion. A suitable pore size may range, for example,from approximately five to 100 microns. Pores may be created bymechanical means or by laser perforation. Pore distribution along aballoon surface may be inhomogeneous to tailor distribution of treatmentagent delivery.

According to any of the embodiments described with reference to FIG. 8and the accompanying text, a treatment agent such as apo A-I mimeticpeptide may be introduced through the inflation cannula (e.g., inflationcannula 830) to expand the balloon (e.g., balloon 825). In the exampleof a balloon of a porous material, such as balloon 825, the treatmentagent will expand balloon 825 and at relatively low pressure (e.g., 2-4atm) diffuse through pores in the porous balloon material to treatmentsite 280 within a lumen of blood vessel 100. FIG. 8 shows treatmentagent 880 diffusing through balloon 825 into a lumen of blood vessel100. Since balloon 825 is positioned at treatment site 285, treatmentagent 880 is diffused at or adjacent (e.g., proximal or distal) totreatment site 285.

The above techniques relate generally to the delivery of a treatmentagent such as apo A-I mimetic peptide through a percutaneous method intoa blood vessel or beyond a blood vessel. Other techniques for deliveringa treatment agent include direct injection into the pericardium orlaparoscopic introduction such as used in bypass surgery or valve repairto a target in the periadventia or myocardium. Surgical deliverytechniques are also suitable and include subxiphoid, periadvential(e.g., at the time of a coronary artery bypass graft procedure) or otherprocedure.

In the preceding detailed description, reference is made to specificembodiments thereof. It will, however, be evident that variousmodifications and changes may be made thereto without departing from thebroader spirit and scope of the following claims. The specification anddrawings are, accordingly, to be regarded in an illustrative rather thana restrictive sense.

What is claimed is:
 1. A composition, comprising: an apolipoprotein A-I(apo A-I) synthetic mimetic peptide complexed with phospholipid; and acarrier associated with the apo A-I synthetic mimetic peptide whereinthe carrier is selected from the group consisting of a liposome, apolymerosome, a micelle, a particle, a gel, a microbubble, aprecipitated peptide particle, a porous glass particle, and a rod. 2.The composition of claim 1 wherein the apo A-I synthetic mimetic peptidecomprises an amino acid sequence or, a C-terminal or N-terminalderivatized amino acid sequence, including one of: (i) an 18-mersynthetic peptide comprising DWFKAFYDKVAEKFKEAF (SEQ ID NO: 1); (ii) an18-mer synthetic peptide comprising DWLKAFYDKVAEKLKEAF (SEQ ID NO: 2);(iii) a 33-mer synthetic peptide comprisingPALEDLRQGLLPVLESFKVFLSALEEYTKKLNTQ (SEQ ID NO: 3); (iv) a 1/9 chimera ofendogenous apo A-I wherein “1” is alpha helix 1 and “2” is alpha helix 2of endogenous apo A-I; and homologs, analogs, and reverse sequences ofeach of (i) through (iv) thereof.
 3. The composition of claim 1 whereinthe phospholipid is one of dimyristoylphosphatidylcholine,1,2-dilauroyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, eggphosphatidylcholine, hydrogenated egg phosphatidylcholine, soybeanphosphatidylcholine, or hydrogenated soybean phosphatidylcholine.
 4. Thecomposition of claim 1 wherein the phospholipid is assembled in amultilamellar vesicle.
 5. The composition of claim 1 wherein a ratio ofapo A-I synthetic mimetic peptide to phospholipid weight to weight isbetween 1:2.5 and 1:5.
 6. The composition of claim 1 wherein thecombination comprises a particle in a range of between 7 nanometers and10 nanometers, the particles subject to aggregation resulting in anaggregate size in a range of between 70 nanometers and 100 nanometers.7. The composition of claim 1 wherein the concentration of apo A-Isynthetic mimetic peptide is between 0.0625 mg/mL to 40 mg/mL.
 8. Thecomposition of claim 1 wherein the apo A-I mimetic peptide is releasedfrom the carrier over a period of time.
 9. The composition of claim 8wherein the period of time ranges from 1 day to 1 month.
 10. A method,comprising: stabilizing an apolipoprotein A-I (apo A-I) syntheticmimetic peptide in solution wherein stabilizing comprises one of: (i)complexing the apo A-I synthetic mimetic peptide with a phospholipid;(ii) increasing a concentration of the apo A-I synthetic mimeticpeptide; or (iii) a combination thereof.
 11. The method of claim 10wherein the apo A-I synthetic mimetic peptide comprises an amino acidsequence or, a C-terminal or N-terminal derivatized amino acid sequence,including one of: (i) an 18-mer synthetic peptide comprisingDWFKAFYDKVAEKFKEAF (SEQ ID NO: 1); (ii) an 18-mer synthetic peptidecomprising DWLKAFYDKVAEKLKEAF (SEQ ID NO: 2); (iii) a 33-mer syntheticpeptide comprising PALEDLRQGLLPVLESFKVFLSALEEYTKKLNTQ (SEQ ID NO: 3);(iv) a 1/9 chimera of endogenous apo A-I wherein “1” is alpha helix 1and “2” is alpha helix 2 of endogenous apo A-I; and homologs, analogs,and reverse sequences of each of (i) through (iv) thereof.
 12. Themethod of claim 10 wherein the phospholipid is one ofdimyristoylphosphatidylcholine,1,2-dilauroyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, eggphosphatidylcholine, hydrogenated egg phosphatidylcholine, soybeanphosphatidylcholine, or hydrogenated soybean phosphatidylcholine. 13.The method of claim 10 wherein the phospholipid is assembled in amultilamellar vesicle.
 14. The method of claim 10 wherein a ratio of apoA-I synthetic mimetic peptide to phospholipid weight to weight isbetween 1:2.5 and 1:5.
 15. The method of claim 10 wherein theconcentration of the apo A-I synthetic mimetic peptide increases from0.125 milligrams per milliliter to 40 milligrams per milliliter.
 16. Themethod of claim 10, further comprising, after stabilizing the apo A-Isynthetic mimetic peptide, associating apo A-I synthetic mimetic peptidewith a bioerodable carrier.
 17. A method of fabrication, comprising:adding a solution containing an apolipoprotein A-I (apo A-I) syntheticmimetic peptide to a solution containing a phospholipid resulting in acombination; and subjecting the combination to a mixing process.
 18. Themethod of claim 17 wherein the apo A-I synthetic mimetic peptidecomprises an amino acid sequence or, a C-terminal or N-terminalderivatized amino acid sequence, including one of: (i) an 18-mersynthetic peptide comprising DWFKAFYDKVAEKFKEAF (SEQ ID NO: 1); (ii) an18-mer synthetic peptide comprising DWLKAFYDKVAEKLKEAF (SEQ ID NO: 2);(iii) a 33-mer synthetic peptide comprisingPALEDLRQGLLPVLESFKVFLSALEEYTKKLNTQ (SEQ ID NO: 3); (iv) a 1/9 chimera ofendogenous apo A-I wherein “1” is alpha helix 1 and “2” is alpha helix 2of endogenous apo A-I; and homologs, analogs, and reverse sequences ofeach of (i) through (iv) thereof.
 19. The method of claim 18 wherein thephospholipid is one of dimyristoylphosphatidylcholine,1,2-dilauroyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, eggphosphatidylcholine, hydrogenated egg phosphatidylcholine, soybeanphosphatidylcholine, or hydrogenated soybean phosphatidylcholine. 20.The method of claim 18 wherein the phospholipid is assembled in amultilamellar vesicle.
 21. The method of claim 18 wherein a ratio of apoA-I synthetic mimetic peptide to phospholipid weight to weight isbetween 1:2.5 and 1:5.
 22. The method of claim 18 wherein thecombination comprises a particle in a range of between 7 nanometers and10 nanometers, the particles subject to aggregation resulting in anaggregate size in a range of between 70 nanometers and 100 nanometers.